Bacteria of the genus Chlamydia are significant pathogens of animals and man. The diseases caused by Chlamydia spp. in man include pneumonia, endocarditis, polyarthritis, blindness, and a wide range of sexually transmitted diseases including cervicitis, salpingitis, pelvic inflammatory disease, and infertility in females and non-gonococcal arthritis and acute epididymitis in males. Despite many years of effort, the Chlamydia remains intractable to genetic analysis due to their obligate intracellular lifestyle and complex developmental cycle. Our long-term goal is to apply the full range of molecular tools including the power of genetics to study the pathogenic mechanisms and intracellular metabolism of Chlamydia. The aims of this application include a technology development aim (genetic tools) and three hypothesis-driven aims to address significant questions of Chlamydia biology. The specific aims are: 1 - Development of tools for the genetic analysis of Chlamydia 2 - Biochemical and genetic characterization of peptidoglycan synthesis in Chlamydia 3 - Identification of transport systems for uptake of essential constituents for Chlamydia growth 4 - Define the Ser/Thr/Tyr protein phosphorylation network in Chlamydia We will employ new and innovative approaches to build on our past success and develop genetic tools in aim 1. Success in achieving this aim will have a significant impact and advance the field of Chlamydia research by making new tools available for genetic analysis of Chlamydia. Aim 2 will identify the enzymes involved in two key cytoplasmic steps in peptidoglycan (PG) synthesis and also apply new techniques to isolate and demonstrate the presence of PG components in Chlamydia. We will also screen for Chlamydia mutants defective in signaling to the host via PG fragments as a first step in dissecting the role of PG in host response. Success in aim 2 will finally resolve the Chlamydia anomaly and reveal potential new targets for antimicrobial development. Aim 3 will characterize transport systems used by Chlamydia to acquire iron and biotin from the host cytosol. Mechanisms of iron acquisition by Chlamydia are completely unknown and our innovative approach will use a Shigella mutant to screen a Chlamydia library. Since the genes for biotin synthesis are absent in C. trachomatis, transport of this essential vitamin is critical for growth. In both cases, success in this aim will shed light on processes of nutrient acquisition that are potential targets for therapeutic intervention. In broader terms, the significance of this aim is that it will provide insight into metabolic processes of obligate intracellular pathogens. Aim 4 is a data generating aim. By defining the Chlamydia phosphoproteome, it will reveal candidate proteins to test for the effect of phosphorylation on protein activity and lead to new hypotheses about how these proteins may regulate differentiation between the dimorphic stages of the Chlamydia developmental cycle.